The Immunogenicity Of Improved Influenza Vlps In Aged Mice

Purpose: Influenza is a highly infectious virus. Each year, seasonal epidemics of influenza cause serious illness and death throughout the world. The aging process in humans is associated with a decrease in immune function (immunosenescence), which can lead to an increased risk and severity of infectious diseases including influenza. The most efficiency strategy to reduce the impact of influenza infections is vaccination. However, the efficacy of influenza vaccines that are licensed for use in the elderly is relatively low (less than 50%) compared to adults (70-90%). To provide better protective measures for the increasingly aging population,new and improved vaccines are needed. Currently, a variety of approaches are being explored to enhance the efficacy of influenza vaccines in the elderly. In the present study, we try to improve virus like particles (VLPs) based influenza vaccines through incorporating of adjuvants and identifying the best routes of vaccine administration.method:In order to improve the immunogenicity of influenza PR8 VLPs in the elderly, we incorporated adjuvants flagellin into influenza PR8 VLPs, and administrated different immunization routes of PR8 VLPs.(1) Flagellin-containing influenza VLPs (Flic-PR8 VLPs) were produced by infection of Sf9 cells with rBVs expressing PR8 HA, M1, NA, and membrane-anchored flagellin. Standard VLPs (PR8 VLPs) containing HA,M1, and NA was also produced. The resulting VLPs were characterized by Western blot analysis, hemagglutination activity analysis, electric microscopic observation, and Cytokine Elisa. (2)Both young and aged mice were immunized twice with 10μg /mouse of Flic-PR8 VLPs and PR8 VLPs which have the equvilant HA activity at 4-week intervals (weeks 1 and 4). For virus challenge, mice were anesthetized with isoflurane and infected with 100 times LD50 of mouse-adapted A/PR/8/34, or 5 times LD50 of California/04/2009 diluted in 50 ul of PBS 4 weeks after the boosting immunization. Blood samples were collected on weeks 0, 2, and 6 by retro-orbital plexus puncture, then the production of HA specific antibody was detected by ELISA, the neutralizing antibody titer was detected by HAI . After challenge, mice were monitored for weight change and signs of illness on a daily basis. Mice that exhibited severe sighs of illness and significant weight loss (>25%) were euthanized accordance with institutional animal care and use committee guidelines. At the end of the challenge study, mice that survived the challenge were sacrificed to collect spleens for analysis of T cell response using ELISPOT method. (3) Both young and aged mice were immunized twice with 2μg/mouse of PR8 VLPs though ID or IM at 4-week intervals. For virus challenge, mice were anesthetized with isoflurane and infected with 100 times LD50 of mouse-adapted A/PR/8/34, or 5 times LD50 of California/04/2009 diluted in 50 ul of PBS. After challenge, mice were monitored for weight change and signs of illness on a daily basis. Mice that exhibited severe sighs of illness and significant weight loss (>25%) were euthanized accordance with institutional animal care and use committee guidelines. At the end of the challenge study, mice that survived the challenge were sacrificed to collect spleens for analysis of T cell response using ELISPOT method.Results:(1)Characterization of VLPs: The Flic-PR8 VLPs are morphologically similar to influenza virions. Characterization of VLPs using Western Blot and HA method showed that both HA and flagellin proteins were incorporated into Flic-PR8 VLPs and the HA protein retained the same functional activities with the PR8 VLPs. Cytokine ELISA result demonstrated that both Flic-PR8 VLPs and PR8 VLPs have the ability to stimulate BMDCs to stimulate cytokine TNF-α. Furthermore, at lower concentration, the Flic-PR8 VLPs was more efficient to stimulate BMDCs, which demonstrated the adjuvant function of flagellin. (2) Immunoresponse post vaccination: Intramuscularly immunization of young with both Flic-PR8 VLPs and PR8 VLPs induces much higher antigen specific IgG in aged mice. However, in the aged group, Flic-PR8 VLPs immunization could induce higher level of IgG compared to PR8 VLPs immunization. Furthermore, we found that flagellin-containing VLPs activated a Th1-preferred Th1/Th2 profile in aged mice, demonstrated by a lower IgG1/IgG2a ratio. Consistant with the ELISA result, HI showed in Flic-PR8 VLPs immunizaed aged mice group,HI titer is 214 versus 128 in PR8 VLPs immunizaed group。(3)Protection rate post challenge: Both PR8 VLPs and Flic-PR8 VLP protected all challenged group against lethal homologous influenza virus challenge. However, only the Flic-PR8 VLP protects old mice from lethal heterotypic influenza virus challenge. (4 )Immunoresponse difference between ID and IM immunization route: In old mice, ID immunization could induce much higher antigen specific IgG than IM immunization. ID immunization activated a Th2-preferred Th1/Th2 profile in aged mice, demonstrated by a higher IgG1/IgG2a ratio. Consistant with the ELISA result, HI showed in PR8 VLPs ID immunizaed aged mice group,HI titer was 2.88 fold higher than IM immunizaced group. (5) Protection rate post challenge: Both IM and ID immunization of PR8 VLPs protected all challenged group against lethal homologous influenza virus challenge. However, only the ID immunization protected old mice from lethal heterotypic influenza virus challenge.Conclusion: (1)Successfully prepared Flic-PR8 VLPs and PR8 VLPs, and they have the same HA activity.(2)In vivo study results showed that Flic-PR8 VLPs has higher immunogenicity in both young and old mice compared to PR8 VLPs(3)Although there is no significant immunogenicity difference between ID and IM route in young adult mice, ID immunization exhibit higher immunogenicity than IM route in old mice.(4)All of the result showed that the immunogenicity of traditional influenza VLPs in aged mice should be improved by incorporation of adjuvants, or by changing the route of vaccine administration.